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Tenth Dr Yellapragada SubbaRow memorial Lecture of
Indra Prastha University, New Delhi
Enzymatic regulation of Lymphaocyte cell fate decisions
and new therapies for human diseases
Shiv Pillai MBBS, PhD, Boston, MA, USA
Dr. Shiv Pillai is Professor of Medicine at Harvard
Medical School and Professor of Health Sciences and
Technology at Massachusetts General Hospital Cancer
Center in Boston, Massachusetts, USA.
After his basic medical education
at Christian Medical College, Vellore, he obtained his
PhD in Biochemistry working in the laboratory of
Professor Bimal Bachhawat initially at Vellore and
later at IICB Kolkatta. As graduate student, he
developed novel chemical cross linking approaches to
study the oligomeric structures of glycoprotein
enzymes. As investigator at the Kothari Center of
Gastroenterology at Kolkatta, he initiated
translational studies on the immunology of amoebiasis.
Moving to USA, he discovered surrogate light chains and
the pre-B cell receptor while postdoctoral fellow at
the laboratory of Prof. David Baltimore of MIT and
In 1988, he was appointed to the faculty at Harvard
Medical School and MGH Cancer Center. He directs
courses in Immunology at Harvard University and Harvard
Medical School. firstname.lastname@example.org
The overriding goal of the work in our laboratory
has been to obtain an in-depth understanding of the
mechanisms involved in the development and activation of B
lymphocytes in order to have insights into pathogenesis
and make new approaches to the treatment of common
Different B lineages develop from distinct haematopoietic
stem cells (HSCs).
B-1 B cells emerge from
fetal liver derived HSCs and B-2 B cells from bone marrow
derived HSCs. Common lymphoid progenitors commit to the B
lineage and initiate rearrangement of immunoglobulin (Ig)
heavy chain gene segments. Developing B cells, which make
in-frame rearrangements of the Ig heavy chain gene,
assemble the pre-B cell receptor and are selected to
survive and expand.
Ig light chain genes
are then rearranged to give rise to immature B
cells with clonal B cell receptors on their cell surfaces
that can engage specific antigens. Immature B cells that
engage self-antigens in the bone marrow with high affinity
are tolerized by a process called receptor editing.
B cells that are not strongly self-reactive exit
the bone marrow and differentiate further in the spleen. A
cell-fate decision at this site contributes to the
generation of two mature B cell subsets - Marginal
Zone B cells that mainly deal with blood-borne pathogens
and Follicular B cells that can collaborate with
T cells and re-circulate through the blood traveling from
lymph node to lymph node in search of antigen and specific
helper T cells.
Protein antigens, typically
from microbes, have both B cell epitopes (“molecular
bumps” that are recognized by antigen receptors on
specific B cells) and internal linear peptides (T cell
epitopes) that facilitate T-B collaboration during an
immune response. T cell help results in the formation of
germinal centers � sites at which events such as isotype
switching and somatic hypermutation occur followed by
selection of high affinity memory B cells and long-lived
B lymphocytes have functions
that go beyond the production of high affinity antibodies
that mediate protection against pathogens. They play
important roles in the activation of specific helper T
cells and in the maintenance of memory CD4+ T cells.
The depletion of B cells using anti-CD20 (Rituxan)
contributes to clinical improvement in a range of diseases
many of which are believed to be actually caused by T
Our discovery of surrogate Ig light
chains and the pre-B cell receptor (the first ever
pre-antigen receptor described) predates by many years the
discovery of its homolog in T cells. We also demonstrated
that the pre-B cell receptor signals constitutively in a
ligand independent manner � not sensing the environment
but monitoring the reading frame. This model is widely
accepted as the mechanism for signaling by both the pre-B
cell and the pre-T cell receptors.
discovered that BTK (Bruton’s Tyrosine Kinase) is
activated downstream of the pre-B cell receptor and the B
cell receptor. BTK inhibitors have been developed by a
number of companies and have recently shown tremendous
promise in both lymphoid malignancies and autoimmunity.
We have also defined a novel “follicular vs.
marginal zone B cell” fate decision in the spleen:
different strengths of BCR/BTK signaling give rise to
follicular B cells or marginal zone B cells.
Our demonstration that canonical NFkB signaling is
required for marginal zone B cell development was the
first report of a developmental role in lymphocytes for
NFkB. We have showed that NFkB and Notch-2 work
synergistically to drive marginal zone B cell development.
We identified two novel stages during B cell
development � a marginal zone precursor (MZP) B cell
development stage and a BTK-independent Follicular B cell
development stage. We have described what we call
Follicular Type II cells. We have also identified a novel
peri-sinusoidal niche in the bone marrow for follicular B
Both B-1 B cells and Marginal Zone B
cells are long-lived self-renewing cells.
Human marginal zone B cells are also known as IgM memory
cells. While we have shown that Notch 2 and NFkB are
required for MZ B cell development, we asked if de novo
DNA methylation is relevant to the self-renewal of
long-lived B cell subsets and B cell memory, especially
since memory lymphocytes display global alterations in DNA
methylation. We conditionally deleted Dnmt3a in
the B lineage.
Young mice displayed a
marked increase of B-1 B cells in their natural habitat �
the peritoneum -- although initial production of B-1 B
cells was not increased. The absence of Dnmt3a
leads to enhanced self-renewal of B-1 B cells. All mutant
mice acquire by the third month the features of a human
monoclonal B lymphocytosis, considered to be a precursor
state of chronic lymphocytic leukemia (CLL). In the Dnmt3a
conditional knockout mice, these expanded B-1 B cells
spill by the fifth month into the blood and spleen
mimicking a full-blown CLL. The leukemic cells closely
phenocopy the more severe version of human CLL in which Ig
genes are largely unmutated.
continue to conduct studies on the evolution of CLL in
this remarkable mouse model looking at global methylation
patterns and using Next Gen Sequencing approaches to
examine changes in B cell repertoires from polyclonal
stages to the clonal leukemia stage. BCR signaling is
important in the disease � in human CLL BTK inhibitors
result in clinical improvement in 90% of CLL subjects.
We have determined that an enzyme, sialic acid acetyl
esterase (SIAE), counters the BTK pathway and is required
for the maintenance of peripheral B cell tolerance and the
prevention of autoimmunity. SIAE regulates inhibitory
signaling in B cells by receptors of the Siglec family.
This enzyme removes acetyl moieties from the 9-OH position
of sialic acid and makes ligands available for inhibitory
receptors of the Siglec family.
In the B
lineage, the absence of this enzyme results in dampening
of inhibitory signals from Siglec2/CD22 and therefore
exaggerates activation of the BCR/BTK pathway.
Hyperactivation of B cells results in a break in
peripheral tolerance and consequent autoimmunity.
A publication in late 2012 from our laboratory follows up
our earlier human studies and clarifies the relevance of
this pathway in human autoimmunity.
B cell have inhibitory signaling molecules like Aiolos,
CD22, and SIAE? We have shown that these inhibitory
pathways exist to “squelch” weakly
self-reactive B cells and prevent them from getting
unwanted T cell help. Such T cell help could drive these B
cells to induce a cytidine deaminase called AID, undergo
somatic hypermutation and become strongly autoreactive.
SIAE and Siglecs prevent promiscuous interactions
between T and B cells.
From a mechanistic
standpoint, phenotypic alterations that occur in mice
lacking SIAE are of biological interest in their own
right. We are currently exploiting these alterations in
seeking novel approaches to both preventive and
is a dramatic enhancement of somatic hypermutation.
Increasing AID levels beyond those seen in activated B
cells does not increase somatic mutation.
However, taking away SIAE causes repeated T-B
interactions, sustains high AID levels and markedly
enhances somatic hypermutation. We are attempting to see
if this finding can be exploited to increase somatic
mutation during preventive immunization against weak but
conserved epitopes of immunogens like gp140 of HIV and our
early results are very promising.
conclusion, many of our fundamental discoveries about cell
fate decisions and BTK signaling have already been
translated into therapies in subjects with autoimmunity
and with B cell malignancies.
companies have developed novel BTK inhibitors that have
proven extremely successful in trials in patients with
After completion of
clinical trials, five BTK inhibitors including Ibrutinib
are now on the market.������
discoveries on the role of the regulation of de
novo DNA methylation in B lymphoid self-renewal are on
the verge of providing novel insights into CLL
pathogenesis and are likely to lead to novel therapies for
CLL beyond BTK inhibitors.
insights into the role of SIAE in regulating T
lymphocyte-B lymphocyte collaboration may well catalyze
novel approaches to enhance somatic hypermutation (for
preventive vaccination) as well as T cell memory (for
obtained into the role of Siglecs and SIAE in controlling
lymphoid versus myeloid differentiation may well translate
into newer therapies in childhood leukemias.� (Based
on Professor Pillai’s summary of his presentation
and his e-mailed clarification)